Fig 2: HNSCC patient PBMCs are able to induce tumor cell killing. PBMCs isolated from (A) 8 healthy controls and (B) 16 HNSCC patients were used in ADCC experiments with VU-SCC-096 cells. PBMCs were harvested after 4 h incubation and analyzed for percentages (%) of CD69 (activation marker) and CD107a (degranulation marker) double positive NK cells (gated as CD3-CD56+ cells). Graphs depict % CD69+CD107a+ NK cells in the presence of cetuximab (CTX, 0.5 µg/mL) or cetuximab in combination with Pam3CSK4 (5 µg/mL). * p < 0.05; ** p < 0.01. (C) Representative flow cytometry dot plots of NK cells stained for CD69 and CD107a of a healthy control in (A) and an HNSCC patient in (B).

Fig 3: Macrophages differentiated with IFN-?3 induce lymphocyte chemotaxis and NK cell activation. Transwell inserts containing autologous PBMCs were placed into wells containing 6-day differentiated M?s ± IFN-?3 to examine immune cell chemotaxis (n = 6). Migrated cells were analyzed by flow cytometry using CD14, CD19, CD3, and CD56 antibodies to identify the number of migrated monocytes, B, NK, and T cells (A). The addition of IFN-?3 to GM-CSF M?s alone, stimulated lymphocyte migration, with significant increases in NK, NKT, and T cell migration (B). Consistent with migration data, GM-M? CCL2, CCL3,CCL4, and CXCL10 secretion were significantly increased by IFN-?3, with no significant effect on M-M?s (C) (n = 7). To assess the role of IFN-?3 on M? activation of NK cells, autologous NK cells were incubated with M?s for 16 h a ratio of 1:1, removed and incubated with K562 target cells for a further 6 h to measure cytotoxicity by degranulation and IFN-? expression (n = 4). GM-M?s differentiated with IFN-?3 significantly increased NK cell cytotoxicity as measured by CD107a expression, as well as IFN-? production (D). Data are representative of two (B,D) and three (C) independent experiments. Paired t-test *p < 0.05, **p < 0.01 (mean ± SE). NE, No expression.

Fig 4: Decreased expression of NK cell-activating receptors on NK cells in JORRP patients. (A) Percentage analysis of NKp30, NKp46, NKG2D, and NKp44 expression on NK cells as determined by flow cytometry of HCs (n = 35) and JORRP patients (n = 35) or different subgroups of JORRP patients. Agg, aggressive (n = 11); Non-agg, non-aggressive (n = 19); 6, HPV6 (n = 14); 11, HPV11 (n = 16); 6&11, HPV6&HPV11 (n = 3). (B) Correlation of CD107a expression on NK cells with NKp30, NKp46, NKG2D, and NKp44 expression on NK cells. Spearman’s correlation coefficients are shown. (C) Correlation of NKp30, NKp46, NKG2D, and NKp44 expression on NK cells with surgical times. Spearman’s correlation coefficients are shown. (D) Correlation of NKp30, NKp46, NKG2D, and NKp44 expression on NK cells with interval time of surgical reoccurrence. Spearman’s correlation coefficients are shown. Data are presented as median with interquartile range. Each dot represents a single patient. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 5: Tumor efficacy and biodistribution of Ad-Cab in syngeneic mouse model CT26 colon carcinoma and xenograft model A549. (A) Schematic diagram of treatment schedule. Mice were treated either with PBS (mock), Ad-5/3?24, Ad-Cab or mPD-L1 after 7 days postengraftment of 5×106 cells in the right flank of the mice. Treatments were given on days 7, 9, 11, 13,17, 19 and 21. Ad-5/3?24 and Ad-Cab were given intratumorally at a dose of 1×109 viral particles while 100 µg of mPD-L1 was given intraperitoneally. (B) Summary data for average tumor growth for all treatment groups for CT26 tumor model. (C) Kaplan-Meier survival curve for the treatment groups. Concentration of His-tagged PD-L1 Fc-fusion peptide in blood (D) and tumor (E) from two mice per each group. Dotted line represents the detection limit of the kit. (F) CT26 tumor free mice were rechallenged with 500,000 CT26 cells and tumor growth was recorded. (G) Schematic diagram of treatment schedule A549 model. Mice were implanted with tumors and human PBMCs and then treated with PBS (mock), Ad-5/3?24 or Ad-Cab. Treatments were given on days 9 and 12 at a dose of 1×109 viral particles intratumorally.(H) Summary data for average tumor growth for all treatments with A549 bearing mice. Concentration of His-tagged PD-L1 Fc-fusion peptide in blood (I) and tumor (J) from two mice per each group. (K) CD8+ T cell and NK cell degranulation (CD107a) and exhaustion (PD-1) markers were examined in the tumor microenvironment. A two-way ANOVA was conducted along with a Dunnett’s test to test significance. The number of mice per each group was 9–10. Levels of significance were set at *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Error bars represent SD. ANOVA, analysis of variance; NK, natural killer; PBMC; peripheral blood mononuclear cell.